Natural Killer (NK) cells are the first line of defense against infection. During their lifetime, NK cells undergo dynamic processes: as they develop, NK cells commit, proliferate, differentiate, and then arrest. Once activated, they immediately acquire cytotoxicity and become quite rapacious in killing target cells. NK cells are predisposed to killing because they are programmed for that function during their development. Our long-term goal is to understand the regulatory mechanisms controlling NK cell dynamics as a prerequisite to the development of successful NK-cell based adoptive immunotherapy. Although the collective efforts, accumulated for the last 20 years, resulted in the identification of key positive regulators of NK cells, the current knowledge failed to identify or elucidate the mechanisms of NK cell negative regulation. We searched for potential candidates and successfully identified TGF? as a potent negative regulator of NK cells1. To date, TGF? remains the only identified negative regulator of NK cell number. Therefore, it is imperative that the mechanisms by which TGF? operate in NK cells are understood. The specific hypothesis driving the proposed research is that TGF? limits the production and restricts the cytotoxicity acquisition of NK cells in response to viral infection. This hypothesis is based on three preliminary data: First, lack of TGF?R signaling results in augmented production of NK cells at terminal differentiation in the bone marrow. Second, lack of TGF?R signaling revealed a previously undescribed population of Granzyme Blow NK cells which is otherwise suppressed in normal mice. Third, mice having TGF?-resistant Granzyme Blow NK cells are resistant to murine cytomegalovirus. Using a combination of in vitro and in vivo approaches, we will test our hypothesis in two Specific Aims: In Aim 1, we will determine the mechanisms by which TGF? controls the production of NK cells. Two studies are designed to investigate i)-how TGF? regulates the processes of differentiation, survival, and proliferation during the production of NK from the bone marrow, and i)-why NK cells at their terminal differentiation are particularly susceptible to TGF? signaling. One prerequisite for the development of successful NK-cell-based immunotherapy in the future is to determine the factors that condition the survival, expansion, and self renewal of NK cells. The possibility that a lack of TGF?R signaling can prolong NK cell survival will support the notion that blocking the TGF?R pathway in NK cells prior to adoptive transfer into patients is a possibility for improving the design of NK-cell-based immunotherapeutic strategies. In Aim 2, we will determine the mechanism by which TGF? restricts functional competence of NK cells during response infection. Three studies were designed to determine direct outcomes of a lack of TGF?R signaling on i)- acquisition of cytotoxicity, ii)-clonal expansion and contraction in response to viral infection, and iii)-tolerance to viral ligands. We believe that understanding how negative regulation by TGF? restricts NK cell cytotoxicity will provide a strategy to make NK cells stronger in facing evolving viruses.